The sensitivity of the improved two-tube test for detection of antimicrobial
residues in Kenyan milk was investigated by comparison with the commercial Delvo
test SP.Suspect positive milk samples (n =244) from five milk collection
centers, were analyzed with the improved two-tube and the commercial Delvo SP
test as per manufacturer, weekly over a ten-week period. The 't' test statistic
was utilised based on the hypothesis: H0: Ud = 0 (U2
­ U1 = Ud) and Ha: Ud >
0 (one-sided test) to analyse the results. The t calculated was compared to
the tabulated t value at p = 0.05 for ten degrees of freedom.The results
suggest that the improved two-tube test has some improved effect on the sensitivity
for antimicrobials residues in comparison to the commercial Delvo test. Utilizing
the improved two-tube test could lead to an improvement in sensitivity for antimicrobial
residues.

Microbiological assays are widely used to detect inhibitory substances in many
countries (Booth & Harding, 1986; Carlsson & Björck 1989; International
Dairy Federation, 1991; Honkanen - Buzalski & Reybroeck, 1995;).The
improved Dutch tube diffusion test is one such method, developed to detect a
broad spectrum of antibiotics at EU MRL levels (Nouws et al, 1995; Rikilt-dlo.,
1998; Nouws et al, 1999;). It involves the inhibition of the test organism,
Bacillus stearothermophilus var. calidolactis if an antibiotic
is present in milk. The test organism is cultured in two tubes in the presence
of nutrients and bromecresol purple as the indicator dye. The pH is modified
to 7.00 and 8.00 to cater for different antibiotics. Under normal conditions
as the culture grows the dye color is changed from purple to yellow. If an antibiotic
is present the culture is killed and the dye remains purple.

In Kenya there is no national control program to ensure milk is free from potentially
harmful drugs, which may be used, for both therapeutic and prophylactic purposes.
This study evaluated whether the improved Dutch tube diffusion test had any
improved effect on the sensitivity for antimicrobials in suspect milk samples
at the European union (EU) maximum residual levels.

Materials and MethodsSampling

Five milk collection centers were randomly selected within the Nakuru district
in the Rift Valley of Kenya. Each center was visited three times weekly for
samples over ten weeks between May and August 2000. Suspect positive samples
from field screening tests in an on going project were tested. Sampling was
done as per IDF standards 50 B, 1995. 244 samples, which were suspect positive,
were tested using the improved two-tube diffusion test and the commercial Delvo
SP test. The analysis was at Egerton University, department of dairy technology,
microbiology section. The raw milk was transported chilled within 2 hrs to the
laboratory. Liquid samples were tested within 8-10 hours after sampling, maintaining
them at not more than 6C between sampling and analysis. The samples were stored
frozen at 20C for further analysis, which was done within one week. Samples
were heated at 80C for 10 minutes in order to eliminate natural inhibitory substances.

Blank control milk, free of antimicrobial substances was collected from the
Egerton University farm Kenya. The milk was stored frozen at -20C and used within
one month. Positive control solutions of the tube diffusion test were prepared
as described by Rikilt.dlo., 1998.

Five mg of pure antimicrobial drugs were weighed and dissolved in 5 ml of the
following reagents: Distilled water (benzylpenicillin), methanol (sulphamethazine);
methanol (dapsone), 0.1 M HCl (oxytetracycline), 0.1 M phosphate buffer pH 8.0
(spiramycin), 0.1 M phosphate buffer pH 8.0 (dihydrostreptomycin). They were
then made up to 100 ml with distilled water. The stock solutions were stored
at -20 C and used within two weeks as per IDF - Group E 503, (1997).
The reagents were purchased from Sigma Chemical Co. (St. Louis, Missouri - USA)
and Merck Darmastadt, Germany to ensure conformity to tested methods. The plate
count agar, Nutrient broth and agar (Difco, Difco Laboratories, Detroit, M1,
USA) were prepared as per the manufacturer and autoclaved at 121 C/15 min. The
test microorganisms Bacillusstearothermophilus var. calidolactis
C 953 spore suspension (107 spores/ml) was supplied by Riklt-dlo
laboratory, the Netherlands and propagated as per Nouws (1995).

Test Procedures

Plate count agar medium was melted and kept at 63C in a water bath. Bromecresol
purple solution (2ml) and B. calidolactis spore suspension (2 ml of 107
spores/ml) was pipetted into the medium (100-ml). The medium was thoroughly
mixed with a final spore concentration estimated at 2 x 105 spores/ml
medium. Chloramphenicol solution 1.5 ml, was added to 100 ml inoculated agar
to prepare medium A and 0.3 ml trimethoprim solution added to 100 ml
inoculated agar to prepare medium B. The mediums A and B were
adjusted to pH 7.0 ± 0.02 and 8.0 ± 0.02, respectively, by 1 M NaOH
solution at 63C. The media was distributed in 1ml portions in test tubes, placed
uprightly and agar let to solidify at room temperature. The prepared test tubes
were used the same day or kept for a maximum of 24 h at 15C.

The suspect milk samples (10 ml of each sample) were pre-heated at 80C for
10 min to inactivate natural inhibitory substances and kill contaminating bacteria.
The samples were then let to cool to room temperature. Phenylbutazone working
solutions (200 ml) was added to each milk sample (10 ml). The final phenylbutazone
concentration was about 400-mg/ml milk. Positive control solutions were prepared
as described by Rikilt-dlo., (1998). The suspect samples (0.33 ml) were pipetted
to tubes A and B, and left at 25 oC for one hour to allow the milk
to diffuse into the medium. After decanting the remaining milk, the tubes with
the positive and negative controls, were all covered with aluminium foil to
protect against dehydration and heat treated in a water bath at 70oC
for 10 min to activate the growth of the spores. The tubes were then incubated
in a water bath at 63oC until the tubes with negative positive control
milk turned yellow and positive control remained purple. This was after 5-6
hrs.

A color index with different levels of resulting colors from yellow to purple
was used to interpret the results. A sample was considered positive if the color
of the tube medium was in agreement with colors 4, 5, 6 of the color index.
A negative result was given if the color of the tube medium was in agreement
with color 1, 2, 3 of the index.

Based on the qualitative colour index the results for the weekly screened suspect
milk from the collection centres using the two-tube test and the commercial
Delvo test were interpreted as in table 1.

From table 1; tcal > 1.833
at p = 0.05, the Ho was rejected and it was concluded that the available
data provided sufficient evidence that the improved two-tube test leads to an
improvement in sensitivity of antimicrobial residues.

The interpretation of the two-tube test is based on a color change of the
medium, which if small can be interpreted either way. 16.4% (n=40) of the samples
were thus classified as inconclusive in the two tube test, since they had passed
the field screening test but failed the two tube on analysis in the laboratory.
26.6 %(n=65) were also inconclusive for the Delvotest. This could be attributed
to the samples having low concentrations of antimicrobials, which on storage
decomposed or false positive results in the screening at the field. The samples
could also contain other inhibitors, which were not sufficient to cause inhibition.
It would be of interest to repeat the study with a more objective test such
as the ELISA.

From the foregoing data the results suggest that the two-tube test has some
improved effect on the sensitivity for antimicrobials residues in comparison
to the commercial Delvo test. The two-tube test could be a suitable method for
a developing dairy industry such as Kenya where previous studies indicate an
urgent need for control of antimicrobial residues at the milk collection centers,
(Shitandi., 2000). The method as developed claims detection limits for beta-lactams,
tetracyclines, sulphonamides, trimethoprim, macrolides and aminoglycosides below
or near the respective MRLs, (Nouws etal, 1995; Rikilt-dlo.,
1998; Nouws et al, 1999;). It offers the advantages of easy performance,
good response to compositional changes, low cost per sample and broad-spectrum
detection.

It is crucial that the hygienic and compositional quality of milk be safeguarded
in the long-term development of a dairy industry. To ensure technological and
toxicological safety, an integrated detection system for antimicrobials must
be developed.

Acknowledgements

The studies were carried out at the Department of Food Science, Egerton University
Njoro- Kenya. I am very grateful to the Swedish Institute (SVENSKA), the scholarship
donor and the Swedish Dairy Training program who kindly provided financial support
during the research period. I am also grateful to Associate Professor Åse
Sternesjö for her devoted guidance throughout the research, Mrs. Lotta
Wall, for excellent technical input. Associate Professor Symon Mahungu, for
permission to use the Egerton University, dairy department facilities for the
local research.

EC-Regulation No. 2377/90 and amendments laying down a Community
procedure for establishment of maximum residue limits of veterinary medicinal
products in foodstuffs of animal origin. Official Journal of the EuropeanCommunities No. L 224, 18.8 1990, p.1.

Rikilt-dlo. 1998 b. Microbiological multi plate system. In the procedure
of the method: Milk and milk products ­ Confirmation of inhibitors. Edition.
1. By Department of microbiology, state institute for quality control of agricultural
products (Rikilt ­dlo), Wageningen, The Netherlands.